Specific Aims The overall goal of our U54 FusONC2 Ewing sarcoma research center is to leverage world-class protein chemistry and structural biology expertise, innovative model systems for functional genomics and small molecule screening to unravel mechanisms by which the EWSR1-FLI1 fusion oncoprotein causes this aggressive sarcoma. That information will enhance our objective of targeting EWSR1-FLI1 activity, with the hope of finding new therapeutics that may improve care for children with Ewing sarcoma. As is described in other sections of this proposal, we are overcoming many of the obstacles barred the development of new drug ? despite knowing of the EWSR1-FLI1 oncoprotein for more than 20 years. Our approach leverages novel experimental systems for functional characterization of EWSR1-FLI1 function in a cellular and organismal context. It is based on a biophysical and biochemical interrogation of EWSR1-FLI1 action, informed by advances our team has made in understanding the structural biology of low complexity domain proteins such as EWSR1. Together these approaches will inform high-throughput chemical screens, with the most promising compounds further refined by medicinal chemistry and focused mechanism of action studies. This effort is strategically situated at the intersection of our team's complementary research expertise in Ewing sarcoma biology, biochemistry and high-throughput screening, and is made possible by the significant institutional resources and track record of UT Southwestern for mechanistically-based cancer drug discovery. Realizing this potential will require careful orchestration of the work accomplished within each of the three Research Projects and the three research Cores. Our capacity to fulfill our longer-term goals will be further enabled by the development and implementation of mechanisms to embrace the larger scientific community, including an External Advisory Committee and other FusOnC2 Centers, as we carry out our research activities. Including other UT Southwestern scientists as well as local and national advocacy groups, can fuel synergies that would not be possible without this Center. Finally, coordinating Center research activities with the educational mission of UT Southwestern holds the opportunity for us to develop the next generation of biomedical researchers focused on Ewing sarcoma and other translocation-driven childhood cancers. Broadly speaking, the goals of the Administrative Core are to ensure effective communications among the investigators, to provide for ongoing evaluation of Center activities, to enable formation of an External Advisory Committee, to integrate our work with the broader cancer biology research community, including other FusOnC2 Centers, and to position the center for effective clinical translation of scientific discoveries that could impact the treatment and outcome for children with Ewing sarcoma.